Regulation of intracellular chloride in hypertension

Abstract

We had previously reported that intracellular chloride (iCl−) is increased in lymphocytes from the spontaneously hypertensive rat (SHR) due to enhanced Cl− influx. The present study examined possible mechanisms of increased Cl− influx in the SHR. iCl− was measured in lymphocytes from SHR and age matched WKY rats 6-10 weeks old using the Cl− sensitive dye, MQAE. The iCl−, assayed in a HCO3− buffered media, was higher in cells from the SHR than those from WKY rats (43±5 vs 26±2 mmol/L, p<0.05). In Cl− depleted cells, re-exposure to Cl− resulted in a rapid influx of Cl− that was faster in the SHR than WKY cells (10.6±0.8 vs 6.9±0.6 mmol/min, respectively (p<0.005). Kinetic analysis of cells exposed to different external [Cl−] revealed that the Vmax was greater in the SHR than WKY (19.8±3.7 vs 12.4±2.0 mmole/min, respectively, p<0.05). There was no difference in the Km for external Cl− (73±7 vs 74±5 mM respectively) or in the Hill coefficients (3.8±0.7 vs 4.6±0.5 respectively). The high Hill coefficients obtained under these assay conditions likely reflects Cl− influx through one or more pathways. Cl− influx in lymphocytes is via a furosemide sensitive Na/K/2Cl co-transporter and through a DIDS sensitive Cl/HCO3 exchanger. We therefore examined these pathways to determine which of these transporters contributed to enhanced Cl− influx in the SHR. Na/K/2Cl co-transport inhibition with furosemide reduced the Cl− influx rates 40-50% and almost completely abolished the difference between SHR and WKY cells (4.9±0.8 vs 4.1±0.9 mmol/min respectively, ns). Cl/HCO3 exchange inhibition with DIDS also attenuated the the Cl− influx rates and almost completely abolished the difference between SHR and WKY cells (5.1±1.8 vs 4.6±1.6 mmol/min respectively, ns). Western blot analysis was used to determine NKCC1 and AE2 protein levels. Neither NKCC1 protein levels (6.13±2.37 vs 5.06±1.93 units, respectively) nor AE2 protein levels were significantly different in the SHR (6.67±2.40 vs 5.02±1.82 units respectively). We conclude that increased Cl− influx through both NKCC1 and AE2 contributes to the increased iCl− in the SHR. Because the protein levels were not different but the Vmax was higher in the SHR, we propose that this may be due to alterations in the post-translational regulation of these transporters.